Role of polymorphonuclear leukocytes in the tumoricidal activity of Propionibacterium acnes

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Virginia Polytechnic Institute and State University

The mechanism responsible for the killing of tumor cells after injection of mice with a mixture of tumor cells and Propionibacterium acnes were investigated. Tumor cells were injected intramuscularly into Balb/c mice either alone or together with P. acnes vaccine. The tumor cells were then removed from the injection site 12 hours after injection, and transferred into fresh mice. Tumor cells from control animals given tumor cells only caused tumors when transferred into fresh mice 12 hours after injection whereas tumor cells from animals given both tumor cells and vaccine did not develop tumors in the fresh nice.

ELISA tests were done to estimate the number of tumor cells in the lesions. In control animals given 10⁵ tumor cells the estimated numbers dropped to 10³ cells at 24 hours, but thereafter rose steadily. Palpable tumors were present 7-10 days later. In animals given 10⁵ tumor cells + 500 ug of P. acnes vaccine, estimated tumor cell numbers fell steadily, and could not be detected after 2 days. Palpable tumors never developed in these animals. These results indicate that tumor cells are killed, or rendered nontumorigenic, during the first 12 hours after co-injection into mice with P. acnes

Histological studies showed that injection of P. acnes vaccine, with or without tumor cells, induced large numbers of polymorphonuclear leukocytes (PMNs) at 12 hours. To determine the role of PMNs in the killing of tumor cells, tumor cells were incubated with supernatant obtained from the phagocytosis mixture of PHNs and P. genes. After a 2- hr. incubation, the tumor cells were washed and injected into fresh mice. No tumors developed, indicating that a product of the phagocytosis of P. acnes by PMNs played a role in the killing of tumor cells. Bacterial vaccines such as P. freudenreichii, which are poorly protective against tuner cells, produced phagocytosis supernatants which were unable to kill tumor cells.

Various oxygen radical scavengers/inhibitors were used to test their effect on the toxicity of the supernatant on tumor cells and chinese hamster ovary cells. Both azide and catalase rendered the supernatant nontoxigenic, suggesting that H₂O₂, produced by PHNs during phagocytosis of P. acnes, is responsible for the killing of tumor cells. However, the addition of catalase 30 minutes after the start of phagocytosis had no effect on the toxicity of the supernatant, suggesting that H₂O₂ is converted to other toxic radicals during the course of phagocytosis of P. acnes by PMNs.

The oxygen consumption levels of PHNs during phagocytosis of P. acnes or other bacterial vaccines was measured and found to be similar regardless of the antitumor ability of the vaccine used. This suggests that the difference in the ability of various vaccines to protect mice against tumors may be in the production of a particular oxygen radical by PMNs during phagocytosis, and not in the production of different quantities of the same radicals.